Dataset Information

Fluorescence detection of GDP in real time with the reagentless biosensor rhodamine-ParM.

ABSTRACT: The development of novel fluorescence methods for the detection of key biomolecules is of great interest, both in basic research and in drug discovery. Particularly relevant and widespread molecules in cells are ADP and GDP, which are the products of a large number of cellular reactions, including reactions catalysed by nucleoside triphosphatases and kinases. Previously, biosensors for ADP were developed in this laboratory, based on fluorophore adducts with the bacterial actin homologue ParM. It is shown in the present study that one of these biosensors, tetramethylrhodamine-ParM, can also monitor GDP. The biosensor can be used to measure micromolar concentrations of GDP on the background of millimolar concentrations of GTP. The fluorescence response of the biosensor is fast, the response time being <0.2 s. Thus the biosensor allows real-time measurements of GTPase and GTP-dependent kinase reactions. Applications of the GDP biosensor are exemplified with two different GTPases, measuring the rates of GTP hydrolysis and nucleotide exchange.

SUBMITTER: Kunzelmann S

PROVIDER: S-EPMC3204873 | biostudies-other | 2011 Nov

REPOSITORIES: biostudies-other

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Fluorescence detection of GDP in real time with the reagentless biosensor rhodamine-ParM.

Kunzelmann Simone S Webb Martin R MR

The Biochemical journal 20111101 1

The development of novel fluorescence methods for the detection of key biomolecules is of great interest, both in basic research and in drug discovery. Particularly relevant and widespread molecules in cells are ADP and GDP, which are the products of a large number of cellular reactions, including reactions catalysed by nucleoside triphosphatases and kinases. Previously, biosensors for ADP were developed in this laboratory, based on fluorophore adducts with the bacterial actin homologue ParM. It ...[more]

PMID: 21812760

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Project description:BackgroundDetection of Acinetobacter baumannii has been relying primarily on bacterial culture that often fails to return useful results in time. Although DNA-based assays are more sensitive than bacterial culture in detecting the pathogen, the molecular results are often inconsistent and challenged by doubts on false positives, such as those due to system- and environment-derived contaminations. In addition, these molecular tools require expensive laboratory instruments. Therefore, establishing molecular tools for field use require simpler molecular platforms. The loop-mediated isothermal amplification method is relatively simple and can be improved for better use in a routine clinical bacteriology laboratory. A simple and portable device capable of performing both the amplification and detection (by fluorescence) of LAMP in the same platform has been developed in recent years. This method is referred to as real-time loop-mediated isothermal amplification. In this study, we attempted to utilize this method for rapid detection of A. baumannii.Methodology and significant findingsSpecies-specific primers were designed to test the utility of this method. Clinical samples of A. baumannii were used to determine the sensitivity and specificity of this system compared to bacterial culture and a polymerase chain reaction method. All positive samples isolated from sputum were confirmed to be the species of Acinetobacter by 16S rRNA gene sequencing. The RealAmp method was found to be simpler and allowed real-time detection of DNA amplification, and could distinguish A. baumannii from Acinetobacter calcoaceticus and Acinetobacter genomic species 3. DNA was extracted by simple boiling method. Compared to bacterial culture, the sensitivity and specificity of RealAmp in detecting A. baumannii was 98.9% and 75.0%, respectively.ConclusionThe RealAmp assay only requires a single unit, and the assay positivity can be verified by visual inspection. Therefore, this assay has great potential of field use as a molecular tool for detection of A. baumannii.

Dataset Information

Fluorescence detection of GDP in real time with the reagentless biosensor rhodamine-ParM.

Publications

Fluorescence detection of GDP in real time with the reagentless biosensor rhodamine-ParM.

Similar Datasets

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